Jul 28, 2023
Microsomal membrane isolation from cell culture
- 1KU Leuven
Protocol Citation: rosanne.wouters, Peter Vangheluwe 2023. Microsomal membrane isolation from cell culture. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbw14vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 31, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82702
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Abstract
Microsomal membrane isolation from cell culture
harvest cells
harvest cells
harvest cells by detachment with TrypLE Express (Gibco, 12604-021)
pellet cells by centrifugation (5 min, 400 gavg, 4°C)
wash cell pellet in phosphate buffered saline (PBS, Sigma, D8537-500ml)
pellet cells by centrifugation (5 min, 400 gavg, 4°C)
lyse cells
lyse cells
resuspend pellet in RIPA lysis buffer (Thermo Scientific, 89900) supplemented with protease inhibitor (Sigmafast, Sigma, S8830-20TAB)
incubate 30min on ice
centrifuge lysate (10min, 100 gavg, 4°C) to pellet and remove nuclear fraction
collect supernatant to continue isolation
isolate microsomal fraction
isolate microsomal fraction
centrifuge supernatant from previous step (10min, 15 000 gavg, 4°C) to pellet the mitochondrial/lysosomal fraction
collect supernatant to continue isolation (pelleted mitochondrial/lysosomal fraction can be stored for future analysis)
centrifuge supernatant from previous step (35 min, 200 000 gavg, 4°C) to pellet the microsomal fraction
resuspend pellet in sucrose (0.25 M)
for Western blotting prepare samples with 20µg protein with 4x LDS loading buffer (NuPAGE, invitrogen, 2399420)